Neutralizing antibodies (NAb) are important in vaccine protection and recovery from viral infection. Thus, understanding the mechanisms governing NAb development could have significant clinical and translational implications. NAbs develop through a process known as affinity maturation in structures known as Germinal Centers (GCs). In GCs, B cells with rearranged immunoglobulin (Ig) genes undergo somatic hypermutation (SHM), leading to enhanced antibody binding to cognate antigens. Ig SHM is primarily mediated by Activation Induced Deaminase (AID), an enzyme that deaminates deoxycytidines into deoxyuridines in transcribing Ig DNA, resulting in G-to-A or C-to-T transitions. Most antibodies have mutations <10% relative to germline, but antibodies that can broadly neutralize global influenza and HIV-1 strains exhibit unusually high SHM levels (up to 33%). Thus, a better understanding of how Ig SHM occurs during viral infections may inform the design of universal vaccines against antigenically-diverse viral pathogens of global importance. Interestingly, the APOBEC3 enzymes are evolutionarily related to AID. APOBEC3 could counteract retroviruses by deaminating reverse transcripts, leading to lethal G-to-A hypermutation. We reported 6 years ago that APOBEC3 encodes Rfv3, a classical resistance gene in mice that promotes recovery from pathogenic Friend retrovirus infection by stimulating a stronger NAb response1. We recently discovered exciting evidence that APOBEC3 could directly edit virus-specific IgG, but in a different sequence context compared to AID2. Thus, we unraveled APOBEC3- mediated deamination as a novel mechanism for antibody diversification in vivo. This fundamental immunological finding unleashed a plethora of basic questions on APOBEC3-mediated Ig SHM, as this process may be a strategy to augment NAb responses during vaccination. In fact, we reported that APOBEC3 can be regulated by IFN? treatment in vivo3. Moreover, we obtained pilot data showing that a TLR7 agonist may augment vaccine IgG responses via APOBEC3. To expand our understanding of APOBEC3-mediated Ig SHM, we therefore propose to: (Aim 1) evaluate regulatory checkpoints for APOBEC3-mediated Ig SHM during viral infection; (Aim 2) investigate the impact of APOBEC3 in vaccine-induced antibody protection; and (Aim 3) determine if increased NAb potency are due to APOBEC3 mutations. Of note, mice encode only 1 APOBEC3 gene, but humans have seven. Thus, the 7 human APOBEC3 proteins may have a stronger impact on Ig SHM. We will utilize a novel transgenic mouse encoding the entire human APOBEC3 locus to test this hypothesis. We will capitalize on our expertise in the Friend retrovirus infection model, utilize novel murine lines specifically generated for the study, investigate clinically-approved vaccine adjuvants and employ innovative nanoparticle vaccines, single B cell PCR and next-generation sequencing approaches. The proposed studies should provide deeper insights on how APOBEC3 mediates Ig SHM that may inform universal vaccine strategies.